Rhus semialata M. extract ameliorate para-phenylenediamine-induced toxicity in keratinocytes

Abstract

para-Phenylediamine (PPD), a major component of hair dyeing ingredients, can induce allergenic sensitization and exert mutagenic, tumorigenic and cytotoxic effect. In this study, we determined the cytotoxic effect of PPD on human keratinocytes and evaluated the protective effect of Rhus semialata M. extracts (RSE) on PPD induced cytotoxicity for the first time. We observed that RSE is a strong inhibitory agent against PPD-induced toxicity in human keratinocytes. The results indicated that RSE pretreatment significantly could suppress PPD induced cytotoxic effects, including decrease of cell viability, accumulation in subG1 phase of cells, and relocation of phosphatidylserine on keratinocytes. Also, we found that PPD caused cytotoxicity was associated with mitochondrial membrane potential loss and subsequent activation of caspase and PARP degradation. However, pretreatment of RSE showed preventive activities against PPD induced mitochondrial membrane potential loss and ROS production in keratinocytes. In conclusion, the results of present study suggest that RSE was able to protect the skin from several cytotoxic effects of PPD and could be a meaningful material in many industries using PPD.

Keywords: Apoptosis; DCFH-DA, 2',7'-dichlorodihydrofluorescein diacetate; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, Dimethyl sulfoxide; DiOC6, 3,3'dihexyloxacarbocyanine iodide; FBS, Fetal bovine serum; Keratinocytes; MTT, 3-[4,5-Dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide; Mitochondrial damage; PI, Propidium iodide; PPD, para-Phenylenediamine; ROS, Reactive oxygen species; RSE, Rhus semialata M extracts; Rhus semialata M; para-Phenylenediamine.

Graphical abstract

Fig. 1

Cytotoxic effect of PPD in HaCaT cells and protective effect of RSE on PPD- induce HaCaT cells. (A) PPD decreased cell viability of HaCaT cells in a dose and time dependent manner. HaCaT cells were treated with various concentration of PPD (0−400 μM) as indicated for 24, 48, and 72 h, repectively. (B) Morphological changes in HaCaT cells were observed using an inverted microscope after treatment of PPD for 48 h. (C) Cell viability was measured by MTT assay. For cell viability analysis, HaCaT cells were seeded on a culture plate, and treated with various concentrations of RSE prior to PPD induction. (D) Cell images of after exposure to PPD with or without RSE treatment for 48 h. The analysis was mean of triplicated measurements ± SD of three separate experiments. *p < 0.05 vs PPD-untreated control, **p < 0.05 vs PPD-treated control.

Fig. 2

PPD induced cell death and protective effect of RSE in HaCaT cells. (A) To determine cell death, HaCaT cells were treated, with or without various concentrations of RSE treatment before PPD induction. Cell cycle distribution was detected by using flow cytometry, after staining of DNA by propidium idodide. (B) Quantification of cell cycle distribution in RSE or PPD treated HaCaT cells. The analysis was mean of triplicated measurements ± SD of three separate experiments. **p < 0.05 vs PPD-treated control.

Fig. 3

Effect of RSE on PPD induced apoptosis in HaCaT cells. (A) Annexin V-FITC and PI staining of HaCaT cells following treatment with RSE prior to PPD induced apoptosis. Cells were incubated with varying concentrations of RSE for 4 h before PPD treatment. After 48 h, the cells were stained with Annexin V-FITC and /or propidium iodide and analyzed by using flow cytometry. (B) Equivalent cells were stained by hoechst 33,342 dye and visualized under the fluorescence microscope. (C) Western blot analysis of caspase-9, caspase-3, cleavage of PARP and β-actin.

Fig. 4

RSE could attenuate the change of ROS and mitochondrial membrane potential (Δψm) after treatment of PPD exposure in HaCaT cells. (A) HaCaT cells were treated with RSE for 4 h, and after that PPD was exposed for 48 h. Cells were stained with 0.1 μM of DiOC₆ for 15 min at 37 °C and loss of Δψm was analyzed by flow cytometry. (B) Changes in DCF fluorescence intensity of HaCaT cells induced by PPD in the presence or absence of RSE pretreatment was measured under a fluorescence microscope. (C) Intracellular ROS levels in HaCaT cells were measured by determination of fluorescence intensity at excitation wavelength of 485 nm and emission wavelength of 535 nm. The analysis was mean of triplicated measurements ± SD of three separate experiments. The analysis was mean of triplicated measurements ± SD of three separate experiments. *p < 0.05 vs PPD-untreated control, **p < 0.05 vs PPD-treated control.

Fig. 5

The chemical components in the extract of Rhus Semialata M. (A) HPLC chromatogram of Rhus Semialata M. extract at 280 nm. (B) Structure of gallic acid and protocatechuic acid.

Fig. 6

Protective effect of gallic acid on PPD- induce HaCaT cells. HaCaT cells were exposed to PPD (200 μM) with or without gallic acid for 48 h. (A) Morphological changes in HaCaT cells were observed using an inverted microscope after treatment of PPD for 48 h. (B) Cell viability was measured by MTT assay. For cell viability analysis, HaCaT cells were seeded on a culture plate, and treated with various concentrations of gallic acid prior to PPD induction. (C) Equivalent cells were stained by hoechst 33,342 dye and visualized under the fluorescence microscope. The analysis was mean of triplicated measurements ± SD of three separate experiments. * p < 0.05 vs PPD-untreated control, ** p < 0.05 vs PPD-treated control.